Exciting New T Cell Therapies Present Unique Challenges in Drug Development.
Immunotherapeutics stimulate the patient's immune system to help fight disease. Most current approaches involve T cells, which are white blood cells that play an important role in the immune process. When functioning properly, T cells are the workhorses of the immune system. They have a critical role in organizing the immune response and killing cells affected by pathogens.
T cells are drug targets themselves for cancer and autoimmune disorders. Now, researchers are re-engineering T cells to help fight cancer and autoimmune diseases. This "re-engineering" is accomplished by removing cells from patients, modifying them, and reintroducing the modified cells to the patient.
Here we provide an overview of these efforts, the unique challenges posed by T cell therapies and engineered T cell development, and capabilities that Fluxion offers to improve these efforts.
T Cells as Targets in Drug Discovery
Checkpoint proteins, such as PD-L1 on tumor cells and PD-1 on T cells, help keep immune responses in check. In certain cancers, the binding of PD-L1 to PD-1 keeps T cells from killing tumor cells. Blocking the binding of PD-L1 to PD-1 with an immune checkpoint inhibitor such as anti-PD-1 allows the T cells to kill tumor cells.
Autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis, and inflammatory bowel diseases. In immune disorders, T cells can be the target of drugs that disrupt the interaction between the T cells and adhesion proteins on vascular endothelial cells.
This prevents a key molecular interaction required for leukocyte adhesion and infiltration, reducing inflammation. For example, natalizumab, approved for treating multiple sclerosis, has shown efficacy in reducing levels of CNS inflammation in MS patients, and is also used for treating Crohn's disease.
Engineered T Cells in Cancer
Cancer immunotherapy using engineered T cells represents an exciting development in cancer treatment. Unlike small molecules or antibodies, cells are living, self-replicating drug delivery vehicles that can be engineered to recognize and kill tumor cells. T cells isolated from cancer patients can be modified to target and kill cancer cells (known generally as adoptive cell therapy, with the most common version being chimeric antigen receptor T cells, or CAR T-cell therapy).
This approach requires drawing blood from patients and separating out the T cells, which are then genetically modified to produce receptors on their surface called chimeric antigen receptors, or CARs. The engineered T cells are expanded in the lab and then re-injected in the patient. Once in the circulation, receptors on the T cells allow the cells to recognize and kill tumor cells expressing the target antigen.
Existing T cell therapies target blood cancers such as ALL, but research is expanding to solid tumor cancers as well. Solid tumor cancers present a greater challenge than blood cells, since the microenvironment that surrounds the tumor cells inhibits the immune response. Nonetheless, research in this area is ongoing.
Engineered T Cells in Autoimmune Disease
In the same way that immune cells can be reprogrammed to target and attack cancer cells, researchers are investigating engineered T cells as a treatment for autoimmune diseases.
In this case, T cells can be engineered with CARs to attack autoreactive immune cells such as B cells. Regulatory T cells, or Tregs, can be extracted from patients and reprogrammed with CARs to "play defense" to suppress immune responses. CAR-modified T cells can be developed that target specific antigens on auto-reactive immune cells and subsequently destroy them.
While this approach holds significant promise, there are many obstacles related to identification of target antigens, cell trafficking, stability, and efficacy that must be overcome before this approach enters the clinic.
Special Challenges in Drug Development
All of these exciting technologies rely on cell-cell interactions, rather than the traditional drug-cell interactions for most therapeutics. In the case of CAR T-cell therapy, the modified cells must modulate their interaction with other cells in the body to produce a therapeutic benefit.
While a variety of high throughput assays have been developed to verify that the drugs can bind to or modify the target T cells, a functional assay is still required to ensure that the T cell produces the desired effect in the human body. As such, studying these cellular interactions in a representative physiological environment is of critical importance in preclinical development.
In traditional drug development, a small molecule or biologic is administered to the patient. Bioavailability, defined as the ability of the therapy to reach the target in the body, is assessed during development using traditional in vitro ADME assays - for absorption, distribution, metabolism, and excretion.
These assays allow characterization and optimization of drug properties prior to moving to lengthy and costly animal studies and clinical trials. These types of tests include physicochemical assays such as solubility and lipophilicity, as well as pharmacokinetic assays such as protein binding, membrane permeability, and CYP450 inhibition.
In the case of engineered T cell therapies, the patient's own T cells are engineered and expanded ex vivo before being re-administered, and this presents unique challenges. Here the bioavailability challenge is different; a main concern is that the T cells have the right properties to achieve efficacy.
Immune cells function by reaching their targets through a complex process that can involve interactions between multiple immune cell types and the target tissue, as well as interactions between the immune cells themselves.
Modeling of this process using an in vitro model requires an assay that can effectively mimic the physiological conditions in the human body. To achieve this, Fluxion has developed the BioFlux "artery on a chip" systems that serve as effective platforms for these functional cell assays. These assays are designed to assess the cell trafficking process, including homing, adhesion, and transmigration.
Cancer cell invasion into the Matrigel matrix.
Time lapse over 15 hours of invasive HT1080 colon carcinoma cells into Matrigel. Cell invasiveness expressed as pixel intensity over total gelled area of the channel as a function of time.
A typical assay in CAR T or drugs targeting T cells will assess adhesion of T cells to an endothelial cell monolayer. Microfluidic BioFlux plates are first prepared by introducing cells into the channels (typically coated with fibronectin, then allowing the cells to grow to a confluent monolayer in an incubator, typically over a period of 24-48 hours. Human vascular umbilical endothelial cells (HUVEC cells) are often used as they are available commercially.
Once the plates are prepared, the treated T cells or CAR T-cells can be added to the plates and then flowed into the channels at physiological shear stress, typically in the range of 1-20 dyn/cm2. Multiple experiments (up to 96 at a time) can be run that vary compound concentration and/or shear stress. The readout for the experiment is made by microscopic imaging, and quantified based on the number of cells adhered or area coverage. Shear stress changes can also be used to measure T cell binding avidity (a quantification of all forces affecting cell-cell binding).
Jurkat cells attached to an activated HUVEC cell. Cells were stained for F-actin and DNA using fluorescently labeled phalloidin (red, focused on HUVEC cells, and green, focused on Jurkats) and Hoescht 33342 (blue) respectively (Scale bar=50 microns).
While this is a commonly-used assay, there are a number of variations that can be employed depending on the anticipated mechanism of action, desired information content, throughput, and cost. Some possible variations on the basic experimental workflow are described below.
Assay Design: What Affects Should Be Measured?
In addition to adhesion, measurements can include rolling velocity, relative change in transmigration through a HUVEC monolayer, or migration/invasion from one cell environment to another (https://www.fluxionbio.com/immunology). All of these effects are measurable with the BioFlux system.
Lymphocytes (in white) migrating through a monolayer of endothelial cells (darker cells) over 8 hours
Other Environmental Variables
In some cases researchers are interested in studying these effects under hypoxic conditions, or differing pH. These variables can be controlled by the BioFlux system.
Endpoint vs. Dynamic Assays
Dynamic assays provide real-time image capture of the cell trafficking process. However, these assays require more time to run the experiments and analyze the data. Endpoint assays are less information-rich, but can be run faster and at lower cost. We find many researchers run the assays in endpoint mode when the number of candidate compounds is still large (hundreds), but switch to dynamic assays when the candidates have been reduced to a manageable number.
Endpoint assay throughput can be further enhanced by the use of the BioFlux Quattro multiplexing system, which allows up to 96 experiments to run simultaneously from a single BioFlux controller.
Cell-Cell Interactions vs. Cell-Protein
Another option to reduce experimental setup time and cost is to run the adhesion studies using an adhesion protein coating rather than a cell monolayer. The coating process takes only minutes and can occur just prior to the experiment, eliminating the 24-48 period required to culture a HUVEC or cancer cell monolayer.
Assay Analysis Options
It is often desirable to build additional analysis options into the experimental design, including protein or gene expression markers in the endothelial layer to test for drug interactions. Media can also be collected after flowing over the endothelial layer for further analysis (PCR, HPLC, LC-MS, etc.).
Run in-House or via Lab Services
More than 500 BioFlux systems are in use globally for applications ranging from basic research to drug development. A variety of systems and protocols are available for running these experiments in your lab. For outsourced projects with fast turnaround, Fluxion also provides lab services to run these assays in our laboratory.
To learn more about BioFlux and its capabilities, contact us on our website.